Competitive Revision application is submitted in response to NOT-OD-09-058, "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications" For the past several years, our laboratory has explored small molecule-nucleic acid conjugates as substrates for an unusual form of organic synthesis in which a small molecule's reactivity is determined by its attached DNA or RNA sequence. We developed the use of these small molecule-nucleic acid conjugates to enable the translation of libraries of DNA templates into corresponding libraries of multistep synthetic small molecules. We subjected the resulting DNA-templated libraries to powerful in vitro selections for target affinity or for bond-forming chemical reaction and identified selection survivors by DNA sequence analysis. These activities have resulted in the discovery of small molecules that inhibit protein targets of current biomedical interest, as well as in the discovery of several new bond-forming chemical reactions. Based on the unusual capabilities of small molecule-DNA conjugates, as well as on the rapidly growing diversity of RNA's known biological functions, we speculated that living systems may be generating and using natural small molecule-nucleic acid conjugates beyond the modest number of known examples (aminoacylated tRNAs, base-modified tRNA and rRNA, and 5'- capped mRNA in eukaryotes). We recently initiated a new line of research within our broader small molecule-nucleic acid conjugate program to develop and apply methods to discover new cellular small molecule-RNA conjugates in a manner that does not depend on any particular small-molecule structure or RNA sequence. These new studies have recently yielded promising initial results including the discovery of two new classes of small molecule-RNA conjugates, demonstrating that the chemical diversity of biology RNA is greater than previously appreciated. In this competitive revision, we propose to identify which RNA sequence(s) are present in the first class of cellular small molecule-RNA conjugates that we discovered, the CoA-linked RNAs. In addition, we will develop a new method to discover these conjugates that is more general that the two methods we currently use, and that in theory can be applied to any small molecule-linked RNA regardless of its chemical lability. Finally, we will characterize our very recent discovery of NAD-linked and lipid-linked RNAs, and we will apply these discovery methods broadly to a variety of prokaryotic, eukaryotic, and Archaean organisms. If successful, these studies will lead to the discovery of new forms of biological RNAs, and may eventually result in the elucidation of new biological pathways for key processes such as regulating gene expression, localizing RNA, or regulating an RNA's lifetime in the cell. Such insights may one day provide new molecular targets for potential therapeutic intervention. PUBLIC HEALTH RELEVANCE: Over the past several decades, the known roles for ribonucleic acid (RNA) in living systems have rapidly expanded. Our proposed work integrates chemistry and biology to discover the first recent examples of new chemical forms of RNA in which RNAs are tethered to small molecules. The resulting discoveries may reveal entirely new biological functions for RNA, illuminate pathways of potential biomedical relevance, and eventually lead to the identification of new therapeutic targets.